During the past year my laboratory established a tissue culture system for the synthesis of subgenomic HCV replicons as originally described by Lohmann and colleagues (Science 286:113, 1999). With the help of this system, we have already gained new insights into host-virus interactions and the antiviral mechanisms that control HCV replication. The scope of our research program is now to exploit this system for a functional analysis of HCV gene products and identification of the host determinants that influence viral replication, including those that are activated by the type I interferon (IFN) response. Our observations that the cell cycle could play a critical role in replication of an RNA virus are, to our knowledge, unprecedented and will provide new insight into host-virus interactions. Furthermore, we will develop procedures to isolate enzymatically active replication complexes from infected cells for the subsequent identification of host factors playing a role in viral replication. Results from these investigations will not only contribute to a better understanding of viral RNA synthesis but also yield new information about host determinants that control tissue tropism and host-range of HCV. Additional experiments are proposed for the investigation of the mechanisms that control the observed resistance of HCV isolates to IFN-a. For this purpose we seek to develop conditions permitting the isolation of IFNresistant viral variants which can then be used for the identification of viral gene products controlling IFN resistance. Apart from our overall approach. we will also establish a detailed genetic and functional map of the viral non-structural region. the availability of which will provide a valuable tool for the successful conduct of the experiments proposed in this application. With the anticipated results obtained from this research program we will not only gain significant information about the mechanism of HCV replication, but will further contribute to the identification of novel targets for antiviral therapy. HCV is a pathogen of global significance that can cause acute and chronic hepatitis and induce hepatocellular carcinoma. Primary HCV infection cannot yet he prevented by vaccination and, so far, effective and affordable treatments to cure the infection in over 170 million carriers is not yet available.